Radio-ligand targeted therapy is a new and promising concept of treatment Castration resistant prostate cancer (CRPC). Only a few radio-pharmaceutics were approved for usage in treating prostate cancer, among the multiple others tested. We aimed to review and summarize the literature on the therapeutic isotopes specific for PSMA.We performed a scoping literature review of PubMed from January 1996 to December 2022.98 publications were selected for inclusion in this review. The studies contained in publications allowed to summarize the data on pharmacokinetics, therapeutic effects, side effects and the medical use of 225Ac and 177Lu radionuclides. The review also presents new research directions for specific PSMA radionuclides.Radioligand targeted therapy is a new and promising concept where Lu-177-PSMA-617 have promising outcomes in treatment according to standard of care. Due to Prostate-Specific Antigen (PSA) testing, PC is diagnosed in early developmental stages when prostatectomy or radiotherapy can be performed with a successfully curative intent. However, in case of metastatic PC, the androgen deprivation therapy is part of the standard care. Nevertheless, in almost all cases, after an average of 2–3 years, the deprivation therapy may generate hormone-resistance, which is defined as the ability of PC to progress even when the testosterone level is at or below the castrate level. Castration-resistant prostate cancer (CRPC) is characterized by poor prognosis, which is reflected in a survival rate <3 years and with limited treatment modalities. In this case, the therapy includes chemotherapy drugs , inhibitors of androgen receptor signaling (ARSI) , inhibitors of polymerase poli-ADP-ribose (PARP), autologous vaccine “Sipuleucel T”, and recently introduced immune checkpoint inhibitors –9. MetasThe development of imaging methods over the years have led to implementation of new ways for PC treatment. Prostate-specific membrane antigen positron emission tomography (PET-PSMA) highlights cancer cells by absorption of radiation from a diagnostic isotope . That isotope connects with a prostate-specific membrane antigen (PSMA) on prostate gland cells or PC cells. Radionuclides therapy uses the abovementioned mechanism by replacing the diagnostic isotope with a therapeutic isotope. This isotope connects with cancer cell antigens (PSMA) and emits beta  or alpha (actinium-225) radiation. Cancer cells yield ionizing radiation with a minor impact on healthy cells. Only a few radio-pharmaceutics were approved for usage in treating prostate cancer, among the multiple others tested. The most recent radionuclide approved by the FDA for PC treatment is 177LuPSMA-617 , 11.The following review aims to discuss and provide information about therapeutic isotopes specific for PSMA.2Glutamine carboxypeptidase II (GCPII), also known as PSMA, has been considered as a potential biomarker of PC, for many years. It is an enzyme coded by FOLH1 gene (folate hydrolase) localized on the short arm of chromosome 11 11p11-p12) 13). GCPI3Currently used ligands, selective to PSMA, may be divided into antibodies and small particles. The small particles are imaging ligands such as MIP-1095, PSMA-617, and PSMA-I&T. The well-known antibodies are 7E11-C5, J591, and PSMA-TTC.The earliest evidence of evaluation of PSMA usefulness for radiotherapy was with 7E11-C5. This molecule targeted an epitope located in the intracellular domain of PSMA. With the help of this antibody, radionuclides of indium-111 and yttrium-90 were made , 22. FurThe radioactive isotopes mentioned above are applicable in modern PC therapy and imaging.4A radionuclide is created by loading radioisotope into the chelator pocket which is bounded to the carrier . They are used in PC treatment after selective binding with PSMA.The chelator is bound to the carrier , and the radionuclide is loaded into the chelator pocket.Small molecules are modified PSMA inhibitors in which the structure of the molecule between the linking region and the radioactive metal chelator DOTA has been altered. Consequently, they do not exhibit homology to antibodies and they possess lower affinity in the nanomolar range than antibodies, so they bind less to PSMA in non-cancerous tissues. When combined with a radioactive element, they exert a cytotoxic effect on cancer cells , 33.Noteworthy, small molecule is PSMA-I&T (imaging and therapy), introduced by Weineisen in 2014 as an additional PSMA ligand . This moAnother, frequently used radioconjugate, known as MIP-1095, is a urea-based small particle designed for the PSMA radiolabeling with iodine I 131 (I131), and it possesses potential antineoplastic activity. Upon the administration of iodine I 131 MIP-1095, the MIP-1095 moiety selectively targets and binds to the extracellular domain of PSMA, thereby delivering cytotoxic iodine I 131 specifically to PSMA-expressing cancer cells .”Antibod5Iodine I 131 was mentioned in early clinical reports about the usage of radionuclides aimed at PSMA. This element was combined with a small particle MIP-1095, as a PSMA ligand. It showed acceptable pharmacological parameters. Its half-life was short and lasted for 8.02 days. Maximal size of the [I131] I-MIP-1095 was 2.4 micrometers, which caused its rapid internalization by cancer cells. Treatment efficacy was satisfactory, as it led to a 50% decrease in PSA concentration in 60% of the cured patients . Current5.1[177Lu] Lu-PSMA-617, also known as Vipivotide Tetraxetan or Pluvictotm are small particle radionuclides approved by FDA for treatment of hormone-resistant PC with metastases. This treatment is suggested for patients who have been treated with inhibitors of androgen receptor and undergone taxan-based therapy . The mos5.1.1177Lu is an isotope which emits radiation in a wavelength between 1–10 millimeters. The emitted energy from the radiation is 479keV , in total. Lutetium is internalized by cells after PSMA binding with the PSMA-617 ligand. PSMA-617 reaches a short cell penetration range till 670 µm. Radionuclide Lu-PSMA-617 is able to generate ionizing radiation affecting only PC cells with a small impact on other cells due to its characteristics. A seven-day half-life of lutetium ensures an optimal therapeutic effect , 41. To 5.1.2Lutetium underwent many clinical trials, which resulted in a wide range of radionuclide treatment results. The most important studies on 177Lu were the phase III VISION and phase II TheraP study.The VISION project measured the effectiveness of 177Lu PSMA-617 treatment on 831 patients with mCRPC. Results of this trial led to the 177Lu PSMA-617 RLT registration by FDA. rPFS  and OS  were assessed for patients treated with the radioligand 177Lu-PSMA-617, after applying SOC (standard of care) compared to patients who received only SOC. Lutetium treatment extended the rPFS up to 7–8 months, compared to that of patients after SOC who had an rPFS of 3–4 months. OS, after applying lutetium, also extended to 15.3 months, compared to 11.4 months after SOC .In the TheraP study, 177Lu PSMA-617 treatment was compared with cabazitaxel therapy on 200 men with mCRPC. Studies measured the frequency of achieved therapeutic effect, which was defined as a 50% reduction in PSA concentration in blood serum compared to the output levels. The mentioned effect was achieved more frequently in cases treated with 177Lu-PSMA-617 than with cabazitaxel, 66% vs. 37% respectively .German Nuclear Medicine Society conducted one of the largest retrospective clinical trials on 177Lu isotope usage. Results showed that use of 177Lu-PSMA-617 in PC treatment causes a significant decrease in PSA by 50% in the blood serum of 65 patients out of 145. Additionally, QoL  rate increased by 60% due to the reduction of pain caused by cancer metastasis to bones .“LuPSMA” conducted by Hoffman et al. is a prospective phase II trial. Results showed that lutetium treatment helped in achieving therapeutic efficacy. It was defined as a 50% reduction in PSA concentration in blood serum, which could be acquired in 57% of all cases. The trial included patients with visceral and lymph node cancer metastasis. Lutetium treatment induced an 82% regression rate of metastatic changes  in this group of patients .OS rate after 177Lu treatment was measured in a few other studies. Violet et al. demonstrate the survival median and OS after lutetium treatment for 13 and 18 months, respectively . Gafity Patients treated with 177Lu-PSMA also experienced a recurrence of the cancer. Recurrence median was 6–9 months. In cases where therapeutic effect of lutetium treatment, defined as a 50% reduction in PSA concentration, was achieved, the recurrence time was 8.3 months. In other cases, the recurrence time was 4.2 months. Other trials were conducted, where patients underwent lutetium nuclide treatment cycle. Therapeutic efficacy was achieved in 26–40% of the patients. Almost half of them regained a stable PSA level in approximately 12 months.In conclusion, the mentioned trials show that 177Lu treatment causes positive therapeutic effects compared to SOC (standard of care) or with cabazitaxel treatment. It effectively extends OS and PFS, along with an increase in QoL.5.1.3Radionuclide treatment may entail some undesired side effects, which must be taken into consideration while making therapeutic decisions. The most frequent side effects of lutetium usage are presented below.5.1.3.1177Lu PSMA-617 accumulated nonspecifically in the salivary glands . Salivar5.1.3.2Hematotoxicity of levels 3–4 after 177Lu PSMA-617 radioligand treatment is the most common side effect. Fourth level thrombopenia appears in 10–46% of all cured patients, anemia in 10%, and fourth level neutropenia in 6–26%. The lowest platelet count, as per morphology tests, was observed after 32 days of lutetium treatment. Lymphocytopenia (level 3–4) was one of the most common signs of hematotoxicity, as it was observed in 32% of the cases.In a German multi-center study, a lesser percentage of anemia, thrombocytopenia, and leukopenia of level 3–4 were observed  . FurtherHematotoxicity, which results from the weak permeability of monoclonal antibodies in solids and their large size accompanied by slow elimination from the body, is a critical problem. Appropriate trials using small molecule PSMA inhibitors to limit this effect are in progress .5.1.3.3Nausea, vomiting, and gastrointestinal disorders usually appear in 50% of the cases, 12 hours after lutetium is given to a patient. It can be caused by an unidentified PSMA expression in the brush border of the proximal part of small intestine, especially duodenum and jejunum. Incidence of this undesired side effect is estimated to be 50%. To avoid this, patients may receive anti-inflammatory and anti-emetic drugs , 58–63.5.1.3.4Fatigue is the most common side effect reported by patients and its incidence is 50% .5.1.3.5PSMA is present in the proximal kidney’s tubules. It leads to nephrotoxicity, as evident in a glomerular filtration rate (GFR) decrease of 11.5 ml after three months of 177Lu treatment Table 1 5.1.4For RLT PSMA treatment, patients should be chosen by a multidisciplinary uro-oncological team, including oncologists, radiotherapists, and urologists, specializing in PC treatment and nuclear medicine specialists.Prostate Cancer Trials Working Group 3 (PCWG3) shows the qualification criteria for treatment : mCRPC w5.1.5There are many clinical trials being conducted around the world. The preliminary findings of 177Lu-PSMA-617 trials are presented below.5.2Isotope 177Lu, connected with small particle PSMA-I&T (imaging and therapy), is another currently used radionuclide. PSMA-I&T is used in imaging diagnostics as well as a carrier for therapeutic ligands in PC treatment.5.2.1In a retrospective study conducted by Heck et al., a 30% decrease in PSA concentration was observed in 56% of cases treated with 177Lu-PSMA-I&T . SimilarBarber et al. compared results of patients treated with 177Lu-PSMA-I&T and who underwent chemotherapy with those of chemotherapy naive patients. For patients who did not receive chemo-therapy, the median of lifetime PFS and OS amounted to 8.8 and 27.1 months and for patients after chemotherapy, it was 6.0 and 10.7 months, respectively .OS rate in a cohort of chemotherapy-treated patients was longer than in the group where the primary treatment was based on taxanes.5.2.2Data showing unwanted side effects of 177Lu-PSMA-I&T treatment are limited. The trial conducted by Heck et al. showed that 9% of the patients suffered from level 3+ anemia, 6% from level 3+ neutropenia, and 4% from level 3+ thrombocytopenia, after 177Lu-PSMA- I&T treatment. Organs which demonstrate the highest amounts of toxicity were the parotid glands and the kidneys Table 2 5.3in vivo half-life, weak tissue penetration, and long blood half-life compared to the small particles aimed at PSMA , but only in a population with known chronic kidney disease . Side ef5.5Results of phase I trial conducted by Tagawa et al. (NCT03276572) are available. They found that after injection of a single dose of 225Ac-J591 radio-therapeutics 41% out of 22 patients experienced more than a 50% decrease in PSA. One patient developed anemia and level 4 thrombocytopenia, after receiving the dose. Further studies on 225Ac-J591 are in progress Table 5 6PSMA 225Ac causes more fractures in double-stranded DNA of cancer cells than those caused by 177Lu. This putative mechanism of action should result in better effectiveness. This strategy guarantees therapy optimization in cases with significant bone metastases. Shorter cumulative tissue explosion on β-radiation preserves the intact bone marrow. However, at the same time, toxicity in salivary glands is noticed. Due to this fact, in some centers it is preferred to administer 177Lu- PSMA to patients with soft tissue lesions, whereas 225Ac- PSMA is injected for high volume bone diseases. In preclinical studies, targeted α-therapy was more successful than therapy with β-emitters. [225Ac] Ac-PSMA-617 also demonstrated a high therapeutic efficacy in patients who experienced progression of PC after 177Lu-PSMA-617 treatment, which shows the high potential to concur resistance before β-emitter therapy , 72, 74.7CTT1403 is an organophosphoramidic peptidomimetic, which binds irreversibly with PSMA, allowing radionuclides to be quickly absorbed into the tumor. Because of its albumin binding component, CTT1403 has a long half-life inside blood, which leads to a higher amount of radionuclides build up around the tumor. CTT1403 reduces tumor mass and improves survival rate of animals with tumor cells transplanted . R2 is a8CRCP is characterized by poor prognosis that is reflected in a poor survival rate and is a terminal disease. Radioligand targeted therapy is a new and promising concept, where a radioisotope is bound with an antibody or a small molecule ligand. The most recent FDA-approved radionuclide used in PC treatment is 177LuPSMA-617. Lu-177-PSMA-617 have promising outcomes in treatment according to standard of care. It effectively extends OS and PFS and increases the QoL. Radionuclide treatment may entail some undesired side effects. The most frequent side effects are xerostomia and hematotoxicity. From limited number of α emitters, 225Ac is the most impressive. Studies showed that PSA was lower in most cases after 225Ac-PSMA-617 treatment. Side effects were mostly limited to xerostomia, xerophthalmia, and weight loss. The most optimal approach seems to be the use of a combination of different radionuclides for maximum therapeutic effect. β− emitters are most commonly used for RLT, which have a low linear energy transfer (LET). The low deposited energy of β− emitters is only able to yield single-strand breaks of the DNA but can perform this over a longer range, which makes them especially suitable for larger tumors. Getting increasingly interesting for RLT, there are alpha emitters, which have a high LET and lower range; therefore, they are more suitable for smaller tumors and micrometastases. Administering alpha emitters at the onset of disease recurrence, when the maximum dose of beta radiation has been reached using 117Lu, could be a beneficial approach . There aPS and PP contributed to the conception and design of the review, drafting and reviewing of the manuscript, revisions/edits, and a review of references. All authors contributed to the article and approved the submitted version.


For the repeatability of the FNPO, SSFSE-DL showed the highest intra-observer  and inter-observer  agreements. The inter-observer 95% limits of agreement (LOA) for SSFSE-DL, SSFSE, and FSE ranged from −3.7 to 4.5, −4.4 to 7.0, and −7.1 to 7.6, respectively. The intra-observer 95% LOA for SSFSE-DL, SSFSE, and FSE ranged from −3.5 to 4.0, −5.1 to 6.1, and −5.7 to 4.2, respectively. The absolute values of intra-observer and inter-observer differences for SSFSE-DL were significantly lower than those for SSFSE and FSE (p < 0.05). Conclusions: Compared with the original SSFSE images and the conventional FSE images, high-resolution SSFSE images with DL reconstruction algorithm can better display follicles, thus improving FNPO assessment.Objective: To investigate the diagnostic performance of high-resolution single-shot fast spin-echo (SSFSE) imaging with deep learning (DL) reconstruction algorithm on follicle counting and compare it with original SSFSE images and conventional fast spin-echo (FSE) images. Methods: This study included 20 participants (40 ovaries) with clinically confirmed polycystic ovary syndrome (PCOS) who underwent high-resolution ovary MRI, including three-plane T2-weighted FSE sequences and slice-matched T2-weighted SSFSE sequences. A DL reconstruction algorithm was applied to the SSFSE sequences to generate SSFSE-DL images, and the original SSFSE images were also saved. Subjective evaluations such as the blurring artifacts, subjective noise, and clarity of the follicles on the SSFSE-DL, SSFSE, and conventional FSE images were independently conducted by two observers. Intra-class correlation coefficients and Bland–Altman plots were used to present the repeatability and reproducibility of the follicle number per ovary (FNPO) based on the three types of images. Results: SSFSE-DL images showed less blurring artifact, subjective noise, and better clarity of the follicles than SSFSE and FSE ( Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, affecting 6–15% of this population . Therefo3 in adults by using transvaginal ultrasonography (TVUS) [According to the revised criteria from the 2003 Rotterdam Consensus Workshop, PCOM was defined as 12 or more follicles at the size of 2–9 mm per ovary and/or OV greater than 10 cmy (TVUS) . The usey (TVUS) ,5,6, noty (TVUS) ,7. The aNot limited by the observation field-of-view, patient size, or sexual history, magnetic resonance imaging (MRI) is increasingly used as an alternative to TVUS in the characterization of the ovarian morphology in both adult women and adolescents with suspected PCOS ,7,8. It FSE imaging is relatively easy to achieve high SNR and spatial resolution at the expense of acquisition time, which may lead to increased motion artifacts. Single-shot FSE (SSFSE), as a fast imaging sequence, is not sensitive to motion, but it is not a preferred sequence for the ovary MRI due to its blurring and relatively low SNR, especially when high-resolution imaging is pursued ,14. ReceThis prospective single-center study was approved by the Clinical Ethics Committee of Renmin Hospital of Wuhan University WDRY2021-K028) and followed the principles of the Declaration of Helsinki. We performed a prior analysis using G. power to determine the required sample size for the comparison of subjective scores of SSFSE with DL or without DL and FSE images. The analysis indicated that a minimum of 34 follicle counts were needed to ensure a test efficiency of ≥0.8. We prospectively recruited a total of 22 clinically confirmed PCOS cases, including 6 adolescent girls and 16 adult women, from the departments of gynecology, endocrinology, reproductive medicine, and pediatrics at Renmin Hospital of Wuhan University between June 2022 and November 2022. The diagnosis of PCOS in adult women met at least two of the following signs: clinically or biochemically confirmed hyperandrogenism, irregular menstrual cycles, and PCOM [021-K028 TM Recon DL, GE Healthcare) was used in the SSFSE sequences (abbreviated as SSFSE-DL), and the original images (abbreviated as SSFSE) were also saved. There were finally three image types, including SSFSE, SSFSE-DL, and FSE, for each participant.All participants underwent ovary MR imaging on a 3-T MR imaging unit  with a 30-channel phased-array coil. The feet-first supine position was adopted for all the participants during the MRI examination. Sedation or injection of contrast agents is not used for good patient compliance during the examination. All MRI protocols consisted of three-plane  T2-weighted FSE sequences  = 352 × 352 and total scan time = 6 min 30 s–7 min 15 s) and matched three-plane T2-weighted SSFSE sequences  = 352 × 352 and total scan time = 1 min 30 s–1 min 54 s). A commercially available DL reconstruction algorithm ; subjective noise on a 3-point scale ; clarity of the follicles on a 5-point scale .The FNPO was determined by counting the number of follicles measuring ≥ 1 mm long axis based on each image type. To avoid a repeat count of the follicles in different slices, the follicles were counted on the axial images, and the corresponding sagittal and coronal images would be referred to at the same time. The assessments of FNPO for both ovaries on each participant were performed twice by observer 1  and once by observer 2 . To avoid any potential recall bias, there was an interval of 4 weeks between two assessments by observer 1.The subjective scoring data were reported as mean ± standard deviation (SD). Qualitative results were compared between SSFSE-DL and SSFSE and between SSFSE-DL and FSE by using Wilcoxon signed-rank tests. Cohen’s weighted kappa was used to assess inter-observer agreement. Kappa values of 0.00–0.20, 0.20–0.40, 0.40–0.60, 0.60–0.80, and 0.80–1.00 indicated poor, fair, moderate, good, and excellent agreement, respectively . Confidet test. A paired t test was also used to compare the repeatability and reproducibility within each image group.Intra-class correlation coefficients (ICCs) were calculated to show the intra-observer and inter-observer agreements for FNPO assessment, including FNPO assessment by reviewer 1 for the first and second session and reviewer 1 and reviewer 2 for the first session. The agreement levels were as follows: ICC greater than 0.80, almost perfect agreement; 0.61–0.80, substantial agreement; 0.41–0.60, moderate agreement; 0.21–0.40, fair agreement; 0.0–0.2, slight agreement; and less than 0.0, poor agreement. Intra-observer and inter-observer variabilities were assessed by Bland–Altman plots . The meap < 0.05 was considered statistically significant.Statistical analyses were performed using MedCalc Software . 2 (range: 16.3–32.2 years). Four adolescent girls admitted no sexual history, and 1 adolescent girl and all 15 adult women had a sexual history. A total of 40 ovaries were included for further evaluation.Among the recruited 22 participants, one adult woman was excluded because of the surgery of the right ovary for ovarian teratoma, and one adolescent girl was excluded due to her intolerance during the MRI examination. Finally, this study included 20 Chinese women (5 adolescent girls and 15 adult women) with a mean age of 22.5 ± 4.7 years (range: 15–31 years), mean gynecological age of 10.5 ± 4.9 years (range: 3–20 years), and mean body mass index (BMI) of 24.2 ± 3.6 kg/mp < 0.05) as well as FSE (p < 0.05). The use of SSFSE helped to reduce the blurring artifacts compared to the FSE sequences, and the application of DL reconstruction helped to reduce the noise of the high-resolution SSFSE images. Two typical cases are shown in Two independent observers conducted the subjective assessment on a total of 40 ovaries in 60 images. p < 0.05). SSFSE-DL showed significantly reduced intra-observer and inter-observer variability for the FNPO assessments. In addition, there were no significant differences between the absolute values of intra-observer and inter-observer differences neither for SSFSE-DL (p = 0.071) nor for SSFSE (p = 0.143). However, for FSE, the absolute values of intra-observer differences were significantly lower than those of inter-observer (p = 0.001) (Bland–Altman plots in the representation of the intra-observer and inter-observer variability of FNPO assessments for each image type are presented in = 0.001) .To the best of our knowledge, this is the first study evaluating the diagnostic performance of DL-reconstructed SSFSE on the antral follicle counts up to the present. Our data demonstrated that SSFSE-DL significantly improved image quality . The counts of the follicles assessed by SSFSE-DL had the best intra-observer repeatability and inter-observer reproducibility for the FNPO assessment.TVUS is currently the most widely used imaging measure to identify PCOM , and thu3 was set for both SSFSE and FSE T2WI sequences in our study. The noise was well resolved in the FSE images at the expense of an acquisition time of approximately 2 min for each sequence. Severe image noise appeared in the SSFSE images on account of the excessively small voxel, let alone its inherently low SNR. The reason is that there is a trade-off between resolution and SNR when using the conventional inverse Fourier transform (iFT) reconstruction method [TM Recon DL. This neural network model embedded in the MR image reconstruction pipeline employs a cascade of over 100 thousand unique pattern recognitions for images with noise and low resolution to reconstruct high-resolution images with high SNR [To achieve high-resolution imaging of the ovary, an identical acquisition voxel of 0.5 × 0.5 × 3 mmn method . In a prn method . Howeverhigh SNR . Better TM Recon DL ahead of conventional reconstruction was one of the main reasons for our result. A conventional image-based linear filter can partially suppress ringing artifacts and noise caused by the iFT reconstruction method, while portions of the acquired k-space data are reduced for consistency with the unsampled high-frequency data, only partially removing these artifacts but broadening the point spread function, thus degrading resolution and blurring images [TM Recon DL can use truncation artifacts as indicators of missing information rather than general suppression of truncation artifacts so as to make the target edge clear to the collected data [In this study, SSFSE-DL images showed fewer blurring artifacts when compared with the original SSFSE and FSE images using conventional reconstruction. The embedded AIRg images . Howeverted data . In addited data . In addited data ,25.3 images were recommended to identify adolescents and young adults with PCOS [The accurate assessment of follicle count plays an important role in the identification of PCOM for patients with suspected PCOS ,27 and iith PCOS . The maxith PCOS . The FNPith PCOS . These v3 on 1.5T, consistencies for the total follicle count were good, while the inter-observer consistency in evaluating follicles 1 to 3 mm in size was poor to moderate [3 on 3T, the intra- and inter-observer consistencies for the total follicle count were substantial, while the inter-observer consistency for follicles 1 to 6 mm in size was moderate [To evaluate the repeatability of follicle counting using the conventional FSE T2WI sequences, various scanning parameters were examined. In an ovarian MRI study with an acquisition voxel of about 0.7 × 0.7 × 4 mmmoderate . In anotmoderate . We specOur study had several limitations. First, only adolescent girls and adult women with clinically confirmed PCOS were included, and no control subjects were included. Moreover, it should be noted that seven of the participants had already received oral hormonal contraceptive agents for treatment prior to imaging, making it infeasible to determine thresholds for PCOM identification in our study. However, our study placed emphasis on the repeatability of the FNPO assessment that was not affected by the inclusion criterion. The identification of PCOM, especially in adolescent girls, using the SSFSE-DL sequence will be explored. Second, the follicles were not classified by size in this study. It was because the blurring artifacts may lead to the unclear delineation of the follicles, resulting in a false measurement of a single follicle or multiple follicles being considered as one. Thus, the classification of follicle size may reduce the reliability of the repeatability assessment of the follicle count, especially when there are severe artifacts. Third, although the follicle counting was performed on the axial images and with reference lines on the other two imaging planes, the respiratory movement and intestinal peristalsis may still lead to a mismatch of the images on the three different planes, potentially resulting in repeated or missed follicle counts. To mitigate the effects of slice mismatch, the incorporation of breath-hold acquisition mode and gastrointestinal motility inhibitors would be an optimal option if possible. Fourth, although the follicles were more clearly displayed on the SSFSE-DL images, the long T2 relaxation time may affect the display of other pelvic structures and the detection rate of lesions, which warrants further investigation.In conclusion, compared with the original SSFSE images and conventional FSE images, high-resolution SSFSE images with DL reconstruction can significantly improve the repeatability and reproducibility of FNPO evaluation, benefiting from the better display of the follicles, which indicates that SSFSE-DL is a more stable and reliable method for follicle count assessment.


Vitamin D is important for immunomodulation and may play a role in autoimmune diseases. Studies have reported a high prevalence of vitamin D deficiency in rheumatoid arthritis (RA) patients, and vitamin D status, assessed by circulating 25-hydroxyvitamin D [25(OH)D] concentration, is inversely associated with RA disease activity. However, it is unclear whether vitamin D deficiency increases the risk of later developing RA. We conducted a systematic review and meta-analysis of pre-diagnostic 25(OH)D concentrations and risk of RA.Medline and Embase databases were searched in December 2021 using various keywords for ‘vitamin D’, ‘rheumatoid arthritis’, and ‘prospective study’. Publications identified from the search were screened for eligibility, studies were excluded if vitamin D status was measured at or after RA diagnosis, and data were extracted from relevant articles. Bayesian meta-analysis was used to estimate the summary relative risk (RR) and 95% credible interval (CrI) for risk of RA in relation to circulating 25(OH)D concentrations, as well as the between-study heterogeneity.I2 = 52%). No associations were evident in men  or women .The search strategy yielded 908 records, of which 4 publications reporting on 7 studies, involving a total of 15,604 participants and 1049 incident RA cases, were included in the meta-analysis. There was no suggestion of an association between 25(OH)D concentration and subsequent risk of RA. The pooled RR per 25 nmol/L increment in 25(OH)D was 0.96 D and RA risk, but there was notable between-study heterogeneity and a lack of precision. Investigations in large-scale prospective studies with long follow-up or suitably designed Mendelian randomisation studies with consideration of potential non-linear relationships are needed to determine whether vitamin D is involved in RA aetiology.The online version contains supplementary material available at 10.1186/s41927-023-00325-y. Rheumatoid arthritis (RA) is a progressive autoimmune disease affecting around 0.5–1% of adults globally . RA maniVitamin D deficiency has been suggested to be associated with various immune-mediated inflammatory diseases including RA –13. ImmuSeveral studies have shown that vitamin D deficiency is more prevalent, and 25-hydroxyvitamin D [25(OH)D] concentrations (which provide a measure of vitamin D status) are lower in RA patients than healthy controls , 16. In An electronic literature search was performed in Medline and Embase databases to identify all eligible studies on the association between pre-diagnostic 25(OH)D concentrations and RA risk that were published from inception to 21 December 2021. The search strategy included keywords and MeSH terms covering a comprehensive list of possible variations of ‘vitamin D’ and its forms, ‘rheumatoid arthritis’, and prospective study designs. The full search strategy for Medline and Embase is listed in Additional File Results from the database searches were merged and duplicates removed using Covidence, a web-based software platform for systematic review management . RecordsData extracted from the eligible papers included: first author name, year of publication, country/location, study name or description, study period, duration of follow-up, number of participants or controls, number of cases, age and sex distribution, study design, outcome assessment, method of 25(OH)D measurement, mean/median 25(OH)D, comparison (the contrast or metric of 25(OH)D), RR/OR/HR and 95% CI, and any adjustments made for covariates. If several multivariable models were reported, the most fully adjusted effect estimate was extracted and used for the meta-analysis. If an article reported pooled results from more than one study, only the individual results were used in this meta-analysis. Data were extracted by one investigator (RC) and separately verified by two investigators (JLC and AKH).The quality of each of the studies included in the meta-analysis was assessed with the Newcastle–Ottawa scale . Each st2, the percent of total variation that is due to true between-study heterogeneity, which is derived from the posterior distribution of τ. The prior distribution for the log RR is weakly informative , based on the assumption that the RR will have a magnitude of less than 3.Prior to meta-analysis, results from all studies were converted to estimates for a 25 nmol/L increment in 25(OH)D. If a study reported results for 25(OH)D in ng/ml, concentrations were first converted to nmol/L by multiplying by 2.496. Given the low prevalence of RA, different effect measures  were considered equivalent to RRs and used directly. If a study reported results separately by sex, or other subgroups, but not overall, the subgroup-specific estimates were pooled using a fixed-effect model before inclusion in the meta-analysis. If a study reported a CI with insufficient precision (due to rounding), the CI was estimated using the reported p-value. Because few studies met the eligibility criteria for inclusion, we used a Bayesian meta-analysis approach to estimate the summary RR and 95% credible interval (CrI) for RA in relation to a 25 nmol/L increment in 25(OH)D concentration. The CrI, similar to a frequentist CI, quantifies the uncertainty around the point estimate. Heterogeneity of RRs across studies is to be expected, given the differences in populations, study designs, and methods for ascertainment of outcomes. A frequentist random-effects model can reliably handle heterogeneity in a meta-analysis of a large number of studies, however, these models are not well-suited to meta-analyses of few studies. A Bayesian approach to meta-analysis accounts for uncertainty in the estimate of heterogeneity inherent in such situations . AdditioTo investigate potential heterogeneity in the results, subgroup analyses were conducted by sex among studies that reported sex-specific estimates. To evaluate whether the results were driven by one large study or a study with an extreme result, a sensitivity analysis was performed sequentially excluding each study from the analysis one at a time and assessing the impact on the summary estimate. As a further sensitivity analysis, frequentist random-effects models were run to estimate the summary RR and 95% CI for risk of RA per 25 nmol/L increment in 25(OH)D. Risk of publication bias was evaluated by visually inspecting funnel plots and using the Egger linear regression test  and BeggAll statistical analyses were performed using R version 4.1.0 , and theThe flow diagram of study selection is shown in Fig. The characteristics of included studies are summarised in Table The average study quality score using the Newcastle–Ottawa scale was 7.7 out of 9 Table . DiffereThe seven studies included in the linear dose–response meta-analysis comprised a total of 15,604 participants and 1049 RA cases. Brink et al.  did not 2 = 52%)  or women   D was 0.96 (95% CrI 0.82–1.13) and there was moderate heterogeneity between studies D ranged from 0.94 (95% CrI 0.77–1.13) when the Nurses’ Health Study (NHS)  was omitP = 0.95) and Begg’s (P = 0.56) tests did not suggest publication bias, but the low number of studies precluded reliable assessment.Visual inspection of the funnel plot D concentration and risk of RA, but the number of studies was small, most studies contained few cases, and estimates were imprecise. Using a Bayesian approach, moderate to high between-study heterogeneity was identified.There is inconsistent evidence for a role of vitamin D in RA development. In agreement with our findings, a Mendelian randomisation analysis did not find evidence of a causal association between 25(OH)D and risk of RA . AnotherAlthough this meta-analysis did not find any association between vitamin D status and RA risk, it is possible that vitamin D plays a role in disease severity and progression. Several studies have reported favourable associations between higher 25(OH)D concentrations or vitamin D supplementation and lower disease activity in patients with RA –18. In aThis systematic review focused on the relationship between pre-diagnostic 25(OH)D concentration and subsequent risk of RA, and thus the findings cannot be directly compared with studies assessing the post-diagnostic association in patient populations. The duration of follow-up (average time between 25(OH)D measurement and diagnosis) in some of the individual studies was only a few years and it is possible that some participants might have had underlying undiagnosed disease when blood samples were collected. An inverse association between 25(OH)D and RA risk was reported among women in NHSII with 25(OH)D measured less than 4 years before symptom onset  increase in 25(OH)D), but not for 25(OH)D measurements 4 or more years prior to diagnosis . SubclinThis systematic review identified very few prospective studies that have investigated the relationship between pre-diagnostic 25(OH)D and risk of RA. The included studies had few RA cases and participants were predominantly women. Moreover, there was a lack of geographical and ethnic diversity, with results available from studies in only three countries , and almost all participants were of European descent. Further research is needed in studies with large numbers of participants that include those of different ethnicities, different geographical locations, broader age groups, and adequate numbers of cases.A limitation of this meta-analysis was the wide variability in assays used to measure 25(OH)D in the individual studies. Measurements of 25(OH)D exhibit considerable inter-assay and inter-laboratory differences, and there is a lack of standardisation of methods, which hinders the ability to directly compare results from studies that report results based on unstandardised 25(OH)D measurements , 38. We Emerging evidence suggests that associations of 25(OH)D with various health outcomes appear to be non-linear, and if vitamin D is beneficial, it is likely to be relevant only for those with vitamin D deficiency , 40. HowA strength of this meta-analysis was the use of a Bayesian approach, which allowed for a more robust estimation of the between-study heterogeneity compared with frequentist methods, which is particularly useful when there are few studies included.In summary, this systematic review and meta-analysis found no association between vitamin D status and risk of RA. Nevertheless, few studies have investigated this relationship, and no firm conclusions can be drawn in the context of limited data. Further large-scale prospective studies with long follow-up durations, a larger number of cases, and enriched with participants with a broad range of 25(OH)D (particularly those with very low concentrations) are warranted to determine whether vitamin D deficiency is associated with the risk of developing RA.Additional file 1. Medline search strategy. Embase search strategy. Figure S1. Frequentist random-effects meta-analysis of 25-hydroxyvitamin D concentration and risk of rheumatoid arthritis. Figure S2. Linear dose-response meta-analysis of 25-hydroxyvitamin D concentration and risk of rheumatoid arthritis, omitting each individual study one at a time. Figure S3. Funnel plot for the meta-analysis of 25-hydroxyvitamin D concentration and risk of rheumatoid arthritis.


Subsequently, a succession of experiments toward this new hydrogel including structure characterization, antimicrobial properties, cell experiment and wound healing were applied. The results of the experiments show that the EPL-TBA hydrogel not only exhibited excellent contact-active antimicrobial activities against Gram-negative bacteria Escherichia coli (E. coil) and Gram-positive Bacteria Staphylococcus aureus (S. aureus), but also inhibited the biofilm formation. More importantly, the EPL-TBA hydrogel promoted the wound healing with low cytotoxicity in vivo. These findings indicate that the EPL-TBA hydrogel has a promising use as a wound dressing in the bacterial infection prevention and wounds healing acceleration.One of the main reasons impeding wound healing is wound infection caused by bacterial colonization with a continuous stage of inflammation. Traditional wound treatments like gauze are being replaced by tissue adhesives with strong wet tissue adhesion and biocompatibility. Herein, a fast-crosslinking hydrogel is developed to achieve both strong antimicrobial properties and excellent biocompatibility. In this study, a simple and non-toxic composite hydrogel was prepared by the Schiff base reaction between the aldehyde group of 2,3,4-trihydroxybenzaldehyde (TBA) and the amino group of  The skin is located on the surface of the body and has the function of sensing the outside world and preventing invasion of the body by external bacteria and pathogens . The occCurrently, hydrogel, the hydrophilic polymers with three-dimensional reticular structure come into view, have both liquid and solid properties . Due to Antimicrobial peptides (AMPs) are a class of small-molecule peptides with functions against external pathogens . AMPs arε-Poly-L-lysine (EPL) is a natural-based peptide with antibacterial properties, and this biological preservative was first used in food preservation in the 1980s. EPL can be broken down in the human body to lysine, which is one of the eight essential amino acids and is allowed to be fortified in food products worldwide [26]. Moreover, EPL has been approved and allowed as a food additive in Japan, Korea, and the United States, and studies have been conducted to confirm that EPL does not cause mutations in bacteria and other pathogens  through Schiff base reaction, and the hydrogel showed promising antibacterial activities  has received some attention due to its antimicrobial properties . MoreoveHerein, we designed a fast cross-linked hydrogel (EPL-TBA) by the Schiff base reaction between EPL and TBA at pH equal to 8.5. In our study, the rapid cross-linking hydrogel were formed within only 5 min. In our study, the physicochemical characteristics like swelling and moisturizing properties of EPL-TBA hydrogels were tested. Antibacterial tests were also conducted on the hydrogel, and showing broad-spectrum antibacterial ability. For biosafety issues, cytotoxicity and hemolysis tests were performed to confirm the excellent biocompatibility of hydrogels. Finally, a wound model using rats was used to demonstrate that EPL-TBA hydrogel aids in the healing process of wounds. Our designed hydrogel can be cross-linked formed in as little as 5 min, and it possesses both excellent antibacterial properties and good biocompatibility, providing a new idea and method for solving wound healing problems in the future.ε-Poly-L-lysine (EPL) (consisting of 25–30 L-lysine residues) was purchased from Zhengzhou Bainafo Bioengineering Co. Ltd. (China). 2,3,4-trihydroxybenzaldehyde, sodium hydroxide, hydrogen peroxide (30%) and Tris-HCl buffer were all purchased from Shanghai Maclin Reagent Co., LTD. Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were derived from The Fifth Affiliated Hospital of Zunyi Medical University. Human skin fibroblast (BNCC337722) were derived from the cell sharing platform of Zhuhai Campus of Zunyi Medical University. DMEM culture medium and fetal bovine serum were derived from Kgi Reagent CD, LTD. NO kit is from Nanjing Jiancheng Bioengineering Institute. IL-6 and TNF-α kits were derived from Jiangsu Enzyme Free Industrial Co., LTD.2O2 (30 wt%) in glass bottles and incubate at room temperature for 5min to form a hydrogel. The hydrogels with different concentrations were named EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80. The 40, 60 and 80 in EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80 refer to the quality concentration of TBA (mg/mL), which equal to the molar concentration 247mM, 371mM and 494 mM of EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80, respectively.The hydrogels were created in a single step using a modified version of a previously reported method . In shor−1 wavenumber range. A UV-vis spectrophotometer  was used to measure the UV-vis spectra of the hydrogels EPL, EPL-TBA-40, EPL-TBA-60 and EPL-TBA-80.To confirm the interaction between EPL and TBA a Fourier transform infrared spectrometer (FT-IR Shimadzu Company of Japan) was employed to analyze the EPL-TBA hydrogels. The FT-IR spectra were captured at 25 °C in the 4,000–400 cmW0) were placed in PBS solutions at different temperatures and removed after a period of time. The weight of the hydrogel (W1) is measured after absorbing the remaining liquid on the surface of the hydrogel with filter paper. The percentage of swelling was then calculated using the Eq.The swelling capacity of different EPL-TBA hydrogels was evaluated by measuring the weight changes before and after the hydrogels in PBS solution . BrieflyW0) were determined. Then they were separately placed in the incubator at 37°C. At predetermined time points, the weight of the hydrogel was measured (W1). The percentage of Water holding ratio was calculated using the Eq.To measure the water retention ratio of EPL-TBA hydrogels, after formation of the hydrogels, the weights of the hydrogels . The rheometer is equipped with parallel plates with a diameter of 25 mm. The frequency scanning range was 0.1–15 Hz, the temperature was set at 37°C, and the constant strain was 1% to test the stability of EPL-TBA hydrogel.S. aureus and E. coli were determined by MIC method (6 (CFU/mL). On a 96-well plate, 50 μL of EPL/TBA in LB  was 2-fold diluted and then mixed with 50 μL of a bacterial culture containing 106 CFU/mL. The final bacterial concentration was 1 × 105 CFU/mL. The OD value of bacteria was determined at OD600nm. If no bacterial growth is observed, this concentration is the MIC value of the bacteria.The antibacterial activities of EPL and TBA against C method . BrieflyS. aureus and MRSA and Gram-negative bacteria E. coli and P. aeruginosa were employed to assess the antibacterial efficacy of the EPL-TBA hydrogel  cells was determined by CCK8. The percentage of cell survival was calculated using the Eq. After incubation with a hydrogel soak, BNCC337722 cells were stained with a LIVE/DEAD staining kit to evaluate the cytocompatibility of BNCC337722 cells. Incubate and stain at room temperature for 30 min, thoroughly clean the samples with PBS, and take photos of the staining results with an inverted microscope.Before being used for 3 days, the EPL-TBA hydrogel was made in a Petri dish, ground into a powder with a grinder, and disinfected with PBS. Around 5,000 human skin fibroblasts (BNCC337722) were plated per well in 96-well plates. In DMEM with 10% (v/v) fetal bovine serum, cells were grown (FBS). At 37°C, cells were grown in a humidified environment with 5% COAs is the absorbance of experimental cells, Ac is the absorbance of control group cells. And Ab is the absorbance of blank group cells.Where, The cells were digested and incubated in 12-well plate. After the cells are filled with each septal hole, the 1 mL gun head was perpendicular to the hole plate to make cell scratches, and the width of each scratch was ensured as far as possible. The cell culture solution was removed, and the orifice plate was rinsed with PBS three times to wash away the cell debris generated by scratches. Add the hydrogel soak solution and culture medium. The culture plates were cultured in an incubator at 37°C, and photographs were taken with an inverted microscope after incubation for 0 h, 12 h, 24 h, and 36 h.S0 is the scratch area on 0 h and S1 is the scratch area on different time.Where, Mouse mononuclear macrophages (RAW264.7) were inoculated in 96-well plates, LPS (5 μg/mL) was used to induce inflammation in the model for 24 h, and EPL-TBA-40 hydrogel of different concentration was added to the 96-well plates, and co-incubated for 24 h. The levels of NO, IL-6, and TNF-αin the supernatant were determined by the Griess reaction and ELISA kit.490nm) of the supernatant. The percentage of hemolysis was calculated using the Eq.The hemocompatibility test was based on a method published by . BrieflyA0 is the absorbance of experimental, A1 is the absorbance of negative control and A2 is the absorbance of positive control.Where, The wound healing assay was modified from . Female S0 is the wound area on day 0, and S1 is the wound area on different days.Where, Wound site tissues were collected at days 3, 7, and 10, fixed with 10% paraformaldehyde solution and embedded in paraffin wax. Tissue sections were stained using hematoxylin and eosin (H&E) staining.All data are expressed as mean ± standard deviation. Two-way analysis of variance (ANOVA) with the Geisser-Greenhouse correction and Tukey’s multiple comparisons test, one-way ANOVA and Holm-Sidak’s multiple comparisons test were used to calculate the significance of differences between groups under test conditions .β-sheet conformation region  and 2,3,4-trihydroxybenzaldehyde (TBA) were picked the main as the main components of hydrogel due to their antibacterial effects on Gram-positive and Gram-negative bacteria. Their excellent antimicrobial ability was demonstrated by MIC assay  by a simple chemical reaction. The Schiff base reaction between the amino groups in EPL and the aldehyde groups in TBA makes the hydrogel fast cross-link in 5 min. The EPL-TBA hydrogels exhibit excellent physicochemical properties. Meanwhile, a series of antibacterial experiments indicated that EPL-TBA hydrogel possesses broad-spectrum antibacterial capabilities and can remove biofilms. Furthermore, EPL-TBA hydrogel has excellent biocompatibility. EPL-TBA-40 hydrogel promoted cell migration while suppressing levels of inflammatory cytokines. More importantly, the full-thickness wound model showed that EPL-TBA-40 hydrogel not only promotes wound healing but also reduces the production of inflammatory cells to promote wound healing. These studies demonstrate that EPL-TBA hydrogel is expected to be a kind of wound dressing, providing a new idea and method for solving wound healing problems in the future.


The study objective was to validate the relative biological effectiveness (RBE) calculated by the modified microdosimetric kinetic model in RayStation (Ray-MKM) for active-energy scanning carbon-ion radiotherapy.The Ray-MKM was benchmarked using a spread-out Bragg-peak (SOBP) plan, which was suggested in literature from the National Institute of Radiobiological Science (NIRS) in Japan. The residual RBE differences from the MKM at NIRS (NIRS-MKM) were derived using several SOBP plans with different ranges, SOBP widths, and prescriptions. To investigate the origins of the differences, we compared the saturation-corrected dose-mean specific energy The benchmark determined the value of the clinical dose scaling factor, This study validated the Ray-MKM based on our active-energy scanning carbon-ion beam via phantom studies. The Ray-MKM could generate similar RBEs as the NIRS-MKM after benchmarking. Analysis based on  Carbon-ion radiotherapy (CIRT) has been used to treat human cancer at the National Institute of Radiological Sciences (NIRS) in Japan since 1994 . Years oTo date, three biophysical models have been used in clinical CIRT. They are the Kanai model , the modversions –16 were versions . The cliIn 2015, our centre started clinical CIRT with an ES beam. Four years later, our centre acquired RayStation . This treatment planning system (TPS) is one of the few commercially available TPSs that can perform MKM-based CIRT. Furthermore, validation of the RBE calculated by the MKM in RayStation (Ray-MKM) was necessary based on our ES beam, but relevant studies were lacking. In the literature, Fossati et al. created and validated a Monte Carlo (MC) dose engine , 19 simuPrior to clinical application, this study examines the Ray-MKM-calculated RBE based on our ES beam via phantom studies. We first benchmark the Ray-MKM by defining 3He-, 12C-, and 20Ne-ion beams  centred at 18.12 cm [range: 21.1 cm (R21.1)] in a virtual water phantom (WP). We then generated a single-beam SOBP plan with a Ray-MKM prescription of 5.8 Gy (D5.8). This plan is abbreviated as R21.1M6D5.8. The dose distributions as well as the RBEs were compared to their counterparts reported by Inaniwa et al. [The benchmark started by configuring the Ray-MKM with the same parameters as the NIRS-MKM. After that, we followed Inaniwa et al.’s approach  to adjusAfter the benchmark, Inaniwa et al. also provided several dose distributions of six cube plans based on their broad beam  to evaluAccording to Eq. , \documementclass2pt{minims in Eq. ,3\documeWe calculated the mentclass2pt{minimIn the literature , 29–32, We first generated various SOBP plans with the Ray-MKM in WPs. The SOBP settings were described as follows: 1, the distance from the centres of SOBPs to the surface of WPs was 7.0 cm (ISO7) and 11.0 cm (ISO11); 2, the shapes of targets were cube and sphere; 3, the diameters or the dimensions of targets were from 4.0 to 12.0 cm with a step size of 2.0 cm; 4, the prescribed Ray-MKM dose was 4.0 Gy; and 5, the beam configurations were single, two opposed, and two orthogonal beams. Then, the LEM was used to recalculate the RBE-weighted dose distributions based on the Ray-MKM-optimized fluence and beam configurations. Finally, the converted LEM doses were compared to existing literature .RBE-Ray) and absorbed (Dabs-Ray) depth dose distributions (DDDs) from the Ray-MKM as well as the respective RBE-weighted (DRBE-NIRS) and absorbed (Dabs-NIRS) DDDs from the NIRS-MKM before (A) and after (B) the benchmark using R211M60D5.8. The local RBE deviation curve, i.e., the black solid lines, ends when the RBE-weighted dose is < 10% of the target prescription. A deviation below that threshold would be neglected since it is not clinically relevant. The deviation of − 3–3% is marked on the figure for reference. The RBE deviation between the Ray-MKM and NIRS-MKM is within 3%. The corresponding target mean dose deviations are listed in Table The mentclass2pt{minimWe further compared the Ray-MKM to NIRS-MKM in terms of different ranges Fig. A and B, Figure mentclass2pt{minimmentclass2pt{minimTable We first benchmarked the Ray-MKM according to the NIRS-MKM based on our ES beam. Then, we validated the residual difference between the Ray-MKM and NIRS-MKM based on an additional six cube plans. The residual errors still existed, and the largest deviation was observed in R15.2M6D5.8. To investigate the origin, we calculated the −1. After years of experience with CIRT, the NIRS was determined to use carbon ions as the reference radiation, and the NIRS-MKM was applied to calculate the RBE for the scanning beam. To build technical continuity with the Kanai model, the NIRS-MKM matched the clinical RBE at the new reference position, i.e., at the centre of R21.1M6D5.8 [The value of s 0.9 Gy . The neuHowever, the different delivery systems could create notable RBE differences. The reformulation of CIRT RBE at the NIRS was based on their broad beam. Fossati et al.’s study  showed twas 2.39 .It seems that the terparts , and theterparts . They coulations . The acculations . Moreoveulations  and GEANulations  were useulations . Thus, dIt is true that the RBE calculation depends on the beam quality and fragment spectra, even when using the same model. However, it seems that the radiation quality of a deep-seated target, i.e., R21.1M6D5.8, can be minimally affected by all of the factors we discussed before. Therefore, the same RBE can be expected. Furthermore, although each facility may have different radiation characteristics and different approaches for simulating the beamline, it is good to determine their centre-specific Compared to the proton beam, the carbon-ion beam has a higher nuclear cross-section when interacting with human tissue, thus creating a central, large angle, low intensity, and non-Gaussian lateral dose halo. The problem created by this halo is that it removes nonneglectable doses from the central Gaussian region. Therefore, accurately modelling the halo is important for predicting either absorbed or RBE-weighted doses. The NIRS-MKM adopted a trichrome approach , 36 for The NIRS treated many more patients than any other single CIRT centre. Their experience was valuable for the whole CIRT community. Therefore, many studies have aimed to convert the NIRS experience, i.e., experience with the Kanai model, to the LEM. As a part of validation, this study confirmed that the Ray-MKM was capable of reproducing the conversion study. However, we should be cautious that the RBE-weighted doses based on even the same absorbed dose between the Kanai model and the NIRS-MKM had a notable difference in the beam entrance and distal end . ConsequThe ’s study . This ta’s study . The conWe validated the Ray-MKM based on our ES beam through several SOBP plans. A benchmark was performed to minimize the RBE difference between the Ray-MKM and NIRS-MKM. To investigate the origin of the RBE difference, we calculated the


P < 0.0001, Yuden’s index = 0.568). The PCR positive rates were estimated as 0.11%, 0.71%, 4.51%, 2.02%, and 13.72%, and PPV was estimated as 0.045, 0.421, 0.951, 0.720, and 0.798 in Periods 1, 2, 3, 4, and 5, respectively. A significant and moderate negative correlation between PCR Ct values and semi-quantitative RADT results was observed . The RADT exhibited good performance in specimens with low Ct values (Ct ≤ 25.00) by RT-PCR. The PPV was significantly higher in Periods 3 and 5, which corresponds to rapid dissemination of the Delta and Omicron variants. The high PPV implies that individuals with a positive RADT result are very likely infected with SARS-CoV-2 and would require prompt quarantine rather than additional RT-PCR testing. The sensitivity of 0.577 indicates that RADT should not replace RT-PCR. Nonetheless, given the high PPV and the ability to track infected persons through rapid results, our findings suggest that RADT could play a significant role in control strategies for further SARS-CoV-2 variants.The COVID-19 pandemic in Korea has dynamically changed with the occurrence of more easily transmissible variants. A rapid and reliable diagnostic tool for detection of SARS-CoV-2 is needed. While RT-PCR is currently the gold standard for detecting SARS-CoV-2, the procedure is time-consuming and requires expert technicians. The rapid antigen detection test (RADT) was approved as a confirmatory test on 14 March 2022 due to rapid dissemination of the Omicron variant. The benefits of the RADT are speed, simplicity, and point-of-care feasibility. The aim of our study was to evaluate the clinical performance of RADT compared to RT-PCR in a single center over 15 months, fully covering the SARS-CoV-2 ‘Variants of Concern (VOC).’ A total of 14,194 cases was simultaneously tested by RT-PCR and RADT from January 2021 to March 2022 in Gangnam Severance Hospital and were retrospectively reviewed. PowerChek SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit, and STANDARD Q COVID-19 Ag Test were used. Positive rates, sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) were estimated for five periods (3 months/period). Receiver operator characteristic curve (ROC) analysis was performed, and Spearman’s rank test assessed the correlation between RT-PCR Ct values and semi-quantitative RADT results. The overall positive rate of RT-PCR was 4.64%. The overall sensitivity and specificity were 0.577 [95% confidence interval (CI) 0.539–0.614] and 0.991 [95% CI 0.989–0.993], respectively. ROC analysis resulted in an area under the curve of 0.786 ( The number of COVID-19 cases escalated in each event, with the appearance of variants of higher transmissibility; therefore, it is necessary to prepare a rapid and reliable diagnostic tool for detection of SARS-CoV-2. Real-time reverse transcription PCR (RT-PCR) has been the gold standard in detecting SARS-CoV-2 due to its high sensitivity and specificity6. Nevertheless, it is time-consuming and requires expert technicians to be performed accurately9. Rapid antigen detection tests could be an alternative option for diagnosis of COVID-19 infection.The World Health Organization declared a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a novel coronavirus disease 2019 (COVID-19), in March 2020. In accordance with global trends, the national situation of COVID-19 in Korea has changed dynamically. The Delta variant was disseminated in early August 2021, and the Omicron variant replaced the Delta variant in December 202110. There have been several studies on the diagnostic performance of RADTs compared with results obtained by RT-PCR; however, most of them were performed with small numbers of samples or were analyzed with inconsistent sample types, reagents, and test kits. Therefore, the clinical performance of RADTs has been reported in a variety of ranges with heterogeneity. In addition, there was a limitation in those prior studies that variants of SARS-CoV-2 could not be fully evaluated14. The aim of our study was to evaluate the clinical performance of the STANDARD Q COVID-19 Ag Test  compared to RT-PCR with a large number of samples over a long period, covering the Alpha, Delta, and Omicron variant pandemics. We also analyzed the diagnostic performance of the STANDARD Q COVID-19 Ag Test according to the change of positive rates of SARS-CoV-2.Initial approval of a rapid SARS-CoV-2 Antigen Detection Test (RADT) was granted in Korea in November 2020. The RADT was approved for use in areas with urgent needs including emergency rooms, intensive care units, and long-term care facilities. The RADT exhibits lower sensitivity in detecting COVID-19 patients compared with PCR-based methods; however, the advantages of RADT are rapidity, simplicity, and point-of-care feasibility with a 15–20 min turnaround timeTo evaluate the clinical performance of RADT comparing with the results of RT-PCR, a retrospective chart review was performed. Inclusion criteria was the cases subjected for detection of SARS-CoV-2 by both RT-PCR and RADT in same day from January 2021 to March 2022. A total of 86,678 samples was tested for detection of SARS-CoV-2 by RT-PCR, and 14,194 cases simultaneously tested by RADT were included in this study  and open reading frame 1ab (ORF1ab) genes of SARS-CoV-2. One drop of 5 μL of extracted template RNA was added to 15 μL of PCR reaction mixture . The indeterminate results were defined when Ct value of only one of the E gene and the ORF1ab gene was below 38 and when Ct values of either E gene or ORF1ab gene was between 38 and 40.RNA was extracted from 300 The STANDARD Q COVID-19 Ag Test was approved by the MFDS of Korea in November 2020. This test is a lateral flow immunochromatographic assay (ICA) that detects the nucleocapsid protein (NP) antigen of SARS-CoV-2 semi-quantitatively. It has two pre-coated lines on the nitrocellulose membrane: control (C) and test (T) lines. A sample of 350 μL of VTM was mixed with the extraction buffer solution, and 2–3 drops of the sample mixture were applied on the sample area of the device. After 15–20 min of incubation at room temperature, results were determined manually with the naked eye. For a positive result, two colored C and T lines were present within 30 min. The test was interpreted as ‘trace’ if the T line was fainter than the C line; ‘1+ (one positive)’ if the T line showed similar intensity to the C line; and ‘2+ (two positive)’ if the T line was thicker than the C line. For a negative result, only the C line was present.P value was less than 0.05.Analyse-itAmong the 14,194 cases, 500 positive and 13,694 negative results were obtained. The positive results consisted of 258 results of 2+, 128 results of 1+, and 114 results of trace , and the number of cases examined using the STANDARD Q COVID-19 Ag Test by period ranged from 1822 to 3217. The gender ratio, median age, PCR qualitative results, and ORF1ab gene PCR, including indeterminate results. Most  of the cases with low CT values under 25.00 gave a positive result with the STANDARD Q COVID-19 Ag Test. In addition, most  of the cases with Ct values under 20.00 showed 2+, and more than half  of the cases with Ct values of 20.00–24.99 also showed 2+ with the STANDARD Q COVID-19 Ag Test. When semiquantitative results of the STANDARD Q COVID-19 Ag Test were compared with Ct values obtained by RT-PCR, a significant and moderate negative correlation was observed   and Youden’s index of 0.568  and 0.991  respectively, which are comparable to the results reported in previous studies18. In addition, three cases of false-negative results in the cases with Ct value < 20. Clerical errors, inappropriate specimen handling, or interference in specimens could be potential reasons19. A total 120 cases (0.89%) of false-positive results were identified in this study. According to the previous report in Canada, the false positive results were identified in 0.05%  of asymptomatic screening tests20, and a false positive rate in another study in Germany was reported to be 0.13% 21. False positive results may lead to unnecessary quarantine and delay of appropriate procedure to patients. The AUC of RADT was suboptimal (0.781) due to relatively low sensitivity and confirmation test with RT-PCR assay should be performed for accurate diagnosis of SARS-CoV-2 infection.There have been previous studies evaluating the clinical performance of RADTs for diagnosing COVID-19 infection. In a German study, 2,028 samples collected from a variable population including patients with COVID-19 symptoms, individuals with SARS-CoV-2 exposure, and hospital staff members showed overall sensitivity and specificity of 42.86 and 99.89%, respectively23. In the German study, higher sensitivities were noted as Ct values < 20, < 25, and < 30 produced results of 100%, 98.25%, and 88.64%, respectively15. Therefore, the RADT could be accurate in diagnosing SARS-CoV-2 infection in the early stage, in which a high viral load is usually identified.Significant correlation was identified between the Ct values obtained by RT-PCR and semiquantitative results by RADT, and the accuracy of RADT was very high, particularly in cases with high viral loads, exhibiting low Ct valuesPPV and NPV are the most important indexes to determine clinical relevance and could vary according to disease prevalence. During the study period, steep increases in prevalence were observed in Periods 3 and 5, which correspond to the Delta and Omicron variant epidemics, respectively. As the prevalence increased, PPVs were confirmed to increase in Period 3 (0.951). The high PPV in the COVID-19 epidemic indicates that an individual with a positive RADT result is highly suspected to be infected with SARS-CoV-2 and should be required to promptly quarantine rather than undergo additional RT-PCR testing. Meanwhile, the PCR positive rate in Period 5 (13.7%) was higher than that in Period 3 (4.5%), but PPV in Period 5 was lower than that in Period 3. During the Period 5, the number of positive cases were dramatically increased up to 4.1% (55/1336), and Ct value of more than half of these cases were ≥ 25.00.24. Prior to the introduction of VOCs, the majority of RADTs was developed with strains that lacked nucleocapsid mutations25, and there have only been a few clinical validation studies that have examined variants affecting antigen test performance. From our results, it can be inferred that the Omicron and Delta variants, which are characterized by many mutations in the spike protein, had little effect on the accuracy of the nucleocapsid targeting RADT. Nonetheless, Omicron and Delta variants also harbor nucleocapsid proteins involving mutations , in conjunction with the spike protein's conspicuous alterations27. While there are some false-negative reports of specimens carrying nucleocapsid mutations that show a high viral load as confirmed by PCR29, it is considered not to affect the accuracy of the RADT since current tests target the C-terminus of the nucleocapsid protein, whereas the great majority of mutations is identified in the N-terminus. In our study, no substantial shift or inclination in specificity was identified according to variant, which indicates the variants did not affect the clinical performance of the RADT. However, we must remain cautious to detect changes in the sensitivity of RADT due to variations with specific mutation trends, and additional studies are necessary to validate this point.Most RADTs target the nucleocapsid protein stably associated with RNA in the virion and is found at higher level than other viral structural proteinsOne of the limitations of this study is that the samples were collected in a single center in a single country; therefore, ethnic or racial diversities could not be considered. In addition, samples tested for pre-hospitalization screening were included; therefore, some asymptomatic patients were inevitably included in our study, which would lower the positive rate. The lack of further investigation about false-positive and false-negative results due to the retrospective study design was another limitation of this study. Finally, STADNARD Q COVID-19 Ag was approved for testing the detection of SARS-CoV-2 by Ministry of Food and Drug Safety of the Republic of Korea in both specimens including nasopharyngeal swab and VTM. Even though the limit of detection of this diagnostic kit in both specimens were validated, possible dilution effect could be occurred when using VTM as a specimen.In conclusion, the STANDARD Q COVID-19 Ag Test showed lower sensitivity compared with RT-PCR; however, this RADT kit showed high PPV in the pandemic situation with a high prevalence of COVID-19 infection, which suggests this RADT kit could be an alternative option for rapid detection of SARS-CoV-2 in a pandemic situation. Further investigation should be performed to compare the results of other RADTs to figure out the clinical usefulness of RADT in diagnosing SARS-CoV-2 infection.Supplementary Information.


The optimized TAC-THQ-NLCs were observed to be smooth and spherical with a particle size of 144.95 ± 2.80 nm, a polydispersity index of 0.160 ± 0.021, a zeta potential of −29.47 ± 1.9 mV, and an entrapment efficiency of >70% for both drugs. DSC and PXRD studies demonstrated the amorphous state of TAC and THQ in the lipid matrix of the NLCs. An FTIR analysis demonstrated the excellent compatibility of the drugs with the excipients without interactions. The TAC-THQ-NLC-based nanogel (abbreviated as TAC-THQ-NG) exhibited a good texture profile and good spreadability. The in vitro release study demonstrated a sustained drug release for 24 h from the TAC-THQ-NG that followed the Korsmeyer–Peppas kinetic model with a Fickian diffusion mechanism. Moreover, the TAC-THQ-NG revealed significantly higher dose-dependent toxicity against an HaCaT cell line compared to a TAC-THQ suspension gel (abbreviated as TAC-THQ-SG). Furthermore, the developed formulations demonstrated antioxidant activity comparable to free THQ. Confocal microscopy revealed improved permeation depth of the dye-loaded nanogel in the skin compared to the suspension gel. Based on these findings, it was concluded that TAC-THQ-NG is a promising combinatorial treatment approach for psoriasis.The aim of this investigation was to develop and analyze a tacrolimus and thymoquinone co-loaded nanostructured lipid carriers (TAC-THQ-NLCs)-based nanogel as a new combinatorial approach for the treatment of psoriasis. The NLCs were formulated by an emulsification-solvent-evaporation technique using glyceryl monostearate, Capryol 90 (oil), and a mixture of Tween 80 and Span 20 as a solid lipid, liquid lipid, and surfactant, respectively. Their combination was optimized using a three-factor and three-level Box–Behnken design (3 Psoriasis is one of the most common autoimmune diseases characterized by chronic skin inflammation. It affects an average of 2 to 4% of the global population . This awStreptomyces tsukubensis, has been widely used for the treatment of psoriasis-like skin disease. It is known to decrease the expression of pro-inflammatory cytokines and, thereby, inflammation [Nigella sativa) seeds. THQ has been found to exhibit outstanding antioxidant, anti-inflammatory, and anticancer effects in both in vitro and in vivo settings. It also exhibits free- and superoxide-radical scavenging action by potentiating the activity of several endogenous antioxidant enzymes [Tacrolimus (TAC), a macrolide immunosuppressant derived from ammation . Howeverammation . Thymoqu enzymes . Further enzymes ,11. Desp enzymes .Several single-drug-based topical formulations are available on the market, but none are able to cure psoriasis effectively. Therefore, combinatorial therapy for better psoriasis management is often advised . The ideStratum corneum (SC) make things worse. This topmost epidermal layer acts as a major impediment to drug administration through the topical route, resulting in limited drug transfer across the skin layers, and, ultimately, lower bioavailability [In addition to the above-mentioned challenges with anti-psoriatic drugs, the presence of the physiological barrier in the form of the lability . The isslability ,19. Amonlability . NLCs nolability . NLCs allability . Anotherlability . Therefore, our study focused on the fabrication of a TAC and THQ co-loaded NLCs (TAC-THQ-NLCs) and an evaluation of its potential efficacy against psoriasis. The developed formulation was optimized using a Box–Behnken design. A nanogel was formulated for ease of application. The developed formulation was characterized in terms of various parameters, such as in vitro drug release, cell culture, and a skin permeation study.2) was observed to be 0.9989 and 0.9998 for TAC and THQ, respectively. The approximate retention time of the TAC and THQ was observed to be 9.9 and 8.0 min, respectively, at their respective wavelengths. The limit of detection (LOD) and limit of quantification (LOQ) for the quantification of the TAC was observed to be 0.065 µg/mL and 0.195 µg/mL, respectively, while for THQ, the LOD and LOQ were observed to be 0.021 µg/mL and 0.062 µg/mL, respectively. A robust, sensitive, and accurate RP-HPLC technique for simultaneous quantification of TAC and THQ was successfully developed and validated. The RP-HPLC chromatogram of TAC, THQ, and a mixture of TAC and THQ is depicted in w/w was chosen for the fabrication of the TAC-THQ-NLCs. As drug solubility in lipids significantly influences the PS, %EE, and drug release of the formulation, it was important to determine drug saturation solubility in the lipids for the development of the NLCs . The solThe stability of the emulsion and its capacity to emulsify were taken into consideration when choosing the surfactants. A higher %transmittance indicates smaller particles, improved physical stability, and non-aggregation of lipid nanoparticles, hence better emulsification. 3-BBD recommended 15 experimental compositions, as shown in X1, X2, and X3) on dependent factors  was quantified and statistically evaluated by using analysis of variance (ANOVA). The regression analysis results for all four dependent factors were fitted independently into different models  to determine the best-fitting model having the highest adjusted and predicted R2, the results of which are summarized in 2” values that were reasonably close to their corresponding “Adjusted R2” values. Three-dimensional surface plots, predicted vs. actual response plots, perturbation plots, and residual vs. run plots obtained for all the dependent factors are shown in The emulsification solvent-evaporation technique was successfully employed to prepare the TAC-THQ-NLCs. This technique of developing NLCs is simple and efficient and can produce particles in the nano-size range. In order to optimize the TAC-THQ-NLCs, 3The PS is one of the most essential quality parameters when it comes to the development of nanoformulations as it not only affects the rate of permeation but also determines how much of the drug is deposited in the skin. In order for a formulation to be effectively delivered to the skin, its particle size should be less than 200 nm . Table 3According to the coded quadratic Equation (1) and various plots as shown in The PDI of the formulation was an essential aspect that not only determined the particle size distribution and homogeneity of the NLCs formulation but also has a substantial impact on their stability. A smaller PDI value indicates that the particles have a narrow size distribution and are homogeneous in nature. Moreover, a PDI value of under 0.3 is desirable as it indicates a uniform distribution of the particles . Table 3According to the coded quadratic Equation (2) and various plots, as shown in Entrapment efficiency is a crucial characteristic in the formulation of NLCs, due to the necessity of delivering the therapeutically effective dose of the drug included in the NLCs. According to the coded quadratic (Equations (3) and (4)) and the various plots shown in The best formulation composition was selected on the basis of set goals i.e., small PS, low PDI, and high %EE. The numerical optimization part of BBD suggested conditions for the TAC-THQ-NLCs preparation as 4.36% lipid-mixture, 6.26% surfactants, and 2.73 min sonication time depending on the applied PS, PDI, and %EE goals, with corresponding predicted value for particle size of 147.98, PDI of 0.164 and %EE of 75.03 and 80.35 for TAC and THQ respectively as shown in The optimized TC-THQ-NLCs were found to have an average particle size and PDI of 144.95 ± 2.80 nm and 0.160 ± 0.021, respectively, as shown in the representative particle size distribution graph in The %EE of TAC and THQ in TAC-THQ-NLCs was found to be 73.44 ± 2.54 and 78.62 ± 2.75, respectively, showing that optimized TAC-THQ-NLCs, displayed higher entrapment of both drugs at the optimum concentration of lipid matrix. The lipophilicity of the drugs and the imperfect crystal structure of NLCs could be the reason for the high entrapment efficiency of drugs in NLCs, as it leads to higher drug payload capacity . FurtherThe morphological characteristics of optimized TAC-THQ-NLCs were investigated under TEM and SEM, and their representative photographs are illustrated in −1 [3 antisymmetric bending), 1356 (CH3 symmetric bending) 1239, 1119 and 1014, 690 (C–H bending) cm−1 [−1 [The FT-IR spectra of the TAC, THQ, GMS, physical mixture , and optimized TAC-THQ-NLCs were recorded, as depicted in −1 . The FT-ng) cm−1 . The FT-cm−1 [−1 . FurtherDSC is a crucial tool for the formulation’s solid-state characterization. It provides information regarding the crystalline or amorphous states of the samples as well as the interaction between the formulation’s components. Following encapsulation in NLCs, the physical state of TAC and THQ was analyzed by PXRD. It determines whether the drugs are present in crystalline or more stable amorphous forms. The diffractogram of TAC, THQ, GMS, physical mixture , mannitol, and lyophilized TAC-THQ-NLCs are displayed in The viscosity and spreadability of TAC-THQ-NG were measured so that a suitable pharmaceutical composition could be made that was consistent, stable, and easy to use. The viscosity was found to be 2.33 ± 0.35 and 2.12 ± 0.28 Pa·s for TAC-THQ-NG and TAC-THQ-SG, respectively, which represents the excellent physical behaviour of the gel. The high viscosity enhances the gel’s residence time on the skin surface, resulting in an increased permeation time. From the perspective of patient compliance, spreadability is a crucial component of topical formulations. The formulation is more comfortable to apply to an inflamed area if the base spreads easily on the skin. The final diameter was found to be 8.10 ± 0.46 cm and 7.23 ± 0.40 cm, respectively, for the TAC-THQ-NG and TAC-THQ-SG, which is indicative of good spreadability. However, the TAC-THQ-SG had lower viscosity and spreadability than TAC-THQ-NG, which might be related to the formulation’s components . The peak force is a measurement of the firmness of the formulation and is indicative of gel strength. The positive area of the curve up to this point measures consistency and it is suggestive of the spreadability of the gel. The maximum negative force indicates the cohesiveness of the sample and is indicative of the extrudability of the sample from the tube. The negative area of the curve is called the ‘work of cohesion’, and it indicates the cohesiveness and consistency/viscosity of the formulation . The forThe cumulative percentage releases of TAC and THQ from TAC-THQ-NG and TAC-THQ-SG were determined in vitro using a dialysis bag over a period of 24 h. The mean percentages of the cumulative drug release of TAC-THQ-NG and TAC-THQ-SG formulations were evaluated and their respective release profiles are shown in 2 value of 0.9892 for TAC, and 0.9822 for THQ from TAC-THQ-NG. Since both TAC and THQ were found to have release exponents (n) of less than 0.5 from TAC-THQ-NG, this is indicative of a Fickian diffusional mechanism for their release.On fitting the in vitro drug release data into different release kinetic models, as shown in 50 values were 0.1303 μgmL−1, 0.876 μgmL−1, 1.528 μgmL−1, and 18.93 μgmL−1, for TAC-THQ-NG, TAC-THQ-SG, TAC-NG, and TAC-SG, respectively, at 24 h of study. The IC50 values showed a higher reduction of hyperproliferative epidermal cells by TAC-THQ-NG compared with that of the TAC-THQ-SG. Compared to free drugs, the increased uptake of the NLCs formulation is responsible for their higher ability to suppress cell proliferation [Psoriasis is characterized primarily by epidermal overexpression with keratinocyte hyperproliferation and differentiation in the epidermis, which produces silvery-scale lesions. Inhibiting epidermal cell proliferation is essential for achieving a more effective therapeutic result in treating psoriasis. Therefore, an MTT assay was performed at 24 h to evaluate the antiproliferative effect of different formulations  on HaCaT cell proliferation, which are depicted in feration . Here, TThere is compelling evidence that free radicals, reactive oxygen species (ROS), the compromised function of antioxidants, and oxidative stress are responsible for the pathophysiology of psoriasis . TherefoThe skin distribution of the TAC-THQ-NG and TAC-THQ-SG was assessed using confocal laser microscopy. The stability data of TAC-THQ-NLCs and TAC-THQ-NG stored at different storage conditions for a period of 3 months are shown in 3-BBD. The optimized TAC-THQ-NLCs showed nanometric-particle-size distribution with good entrapment efficiency. An in vitro anti-proliferation study showed a dose-dependent effect on the HaCaT cells (keratinocyte line) with TAC-THQ-NGs showing higher efficacy than their suspensions. An in vitro drug release study showed a sustained release of TAC and THQ for 24 h from the TAC-THQ-NG. Moreover, the depth of skin permeation measured by CLSM revealed higher permeation of the TAC-THQ-NG as compared to that of the TAC-THQ-SG. The above results proved the potential of the optimized TAC-THQ-NLCs for not only improving the treatment of psoriasis but at the same time reducing dose-related toxicity, which needs to be validated by conducting in vivo studies.Psoriasis is a chronic skin condition that impacts patients both physically and socially. Patients need to undergo treatment on a regular basis for many years to avoid recurrence. Monotherapies often fail to treat the disease effectively. Hence, it is very important to deliver multiple drugs to the inner-skin tissues. Here, TAC-THQ-NLCs were formulated using an emulsification solvent-evaporation technique and optimized using 3Tacrolimus (TAC) was received as a gift sample from Concord Biotech, Gujarat, India. Thymoquinone (THQ) and Rhodamine B were purchased from Sigma Aldrich, Saint Louis, MO, USA. Capryol 90, Labrasol, Gelot 64, Apifil CG, Precirol ATO 5, Labrafil M 1944 CS, Lauroglycol 90, Peceol, Masine 35-1, and Plurol Diisostearique were obtained from Gattefose India Pvt. Ltd., Mumbai, India as a gift sample. Triethanolamine, stearic acid and glyceryl monostearate  were procured from CDH Fine Chemicals . (Span 80 and 20), Mannitol and Tween 80 were purchased from SD Fine Chemicals . PEG 400 was procured from Thomas Baker Chemicals, New Delhi, India. Lubrizol India  was kind enough to gift the Carbopol Ultrez 10 NF for this work. Analytical-grade acetonitrile and methanol were procured from Qualigens Fine Chemicals, Mumbai, India. The remaining chemicals and reagents used for this research work were of analytical grade.v/v, respectively. The sample volume was kept at 20 μL with a total run time fixed for this elution process at 20 min. Prior to analysis, the samples were degassed and passed through a (0.2 µ) nylon filter. Finally, the concentrations of TAC and THQ were measured at wavelengths of 210 nm and 254 nm, respectively.The concentration of TAC and THQ in the developed NLCs was simultaneously measured by our developed RP-HPLC  technique. For chromatographic separation of drugs, RP-HPLC apparatus with a C-18 column  was used, equipped with a photodiode array (PDA) detector. Acetonitrile and water were used in binary combination as the mobile phase; at a flow rate and ratio of 0.6 mL/min and 90:10, For developing the TAC-THQ-NLCs, the lipids were selected based on the saturation solubility of drugs. For selecting the liquid lipid (oil), different liquid lipids in 1 mL quantities were transferred into micro-centrifuge tubes (MCT), and an excess quantity of the drug was gradually added and dissolved using a mechanical shaker  at room temperature for 24 h. Then, the samples were centrifuged for 0.5 h at 5000 rpm at room temperature . Thereafter, the supernatant from each tube was collected and dissolved in methanol, filtered with a membrane filter (0.2 µ), and the saturation solubility of the drug was quantified spectrophotometrically  . For selv/v) was added into the organic phase containing lipids mixture under constant stirring. Next, the organic solution was evaporated by heating at 40 °C for 3–4 h on a digital magnetic stirrer. The resulting solution was further diluted 10 times using Milli-Q water, and the %transmittance was noted spectrophotometrically at 510 nm wavelength. The surfactant which revealed the highest %transmittance was considered the best for developing the NLCs [For the successful development of the NLCs, the surfactants were selected based on their emulsification capacity of lipid mixture. Hence, for selecting the best surfactant, a lipid mixture (100 mg) was dissolved in 3 mL dichloromethane, and the aqueous solution of different surfactants solution  were prepared using the previously described emulsification solvent-evaporation technique with slight modifications ,50. Brie® software V.11.0 . For the optimization study, different codes were assigned for the independent as well as the dependent factors. Codes for the independent factors were assigned as follows: “X1” for lipid mixture (3–5% w/v), “X2” for surfactant mixture (5–7% w/v), and “X3” for sonication time (2–4 min.) while codes for dependent variables were chosen as: “Y1” for particle size (nm), “Y2” for PDI, “Y3” and “Y4” for entrapment efficiency of TAC and THQ (%EE). The above-mentioned parameters with their respective values are summarized in 3-BBD provides 15 different combinations, including 3-center points (repetitions) for the development of the NLCs. Each formulation, as per the composition, was prepared to obtain the results, which were later fitted into the design as given in For the development of NLCs with desired physicochemical characteristics, three factors  and three levels  based on the Box–Behnken design were selected for the optimization of our developed formulation using Design-ExpertThe particle size (PS), polydispersity index (PDI), and zeta potential (ZP) of the developed TAC-THQ-NLCs were measured by a zeta sizer  by the principle of dynamic light scattering. For analysis, the formulations were diluted 20 times with deionized water and then analyzed at a 90° angle of scattering at room temperature. Similarly, for the determination of ZP, the same samples were placed in the electrophoretic cell before analysis . All theThe %EE and %DL of the TAC-THQ-NLCs were determined indirectly by the ultracentrifugation technique. Briefly, the formulations were transferred to a centrifuge tube and subjected to high-speed centrifugation  at 15,000 rpm for 15 min. After that, the supernatant was collected, diluted appropriately with methanol, and filtered with a 0.2 µ membrane filter. After that, the concentration of unencapsulated drugs was quantified by RP-HPLC . Finallyw/v) to enhance the contrast. Finally, the sample was air-dried at room temperature before being examined under TEM  operated at 200 kV with point-to-point resolution. For SEM analysis, 20 µL of the formulation was placed on the aluminum stub-bearing double-sided tape. Thereafter, the formulation-bearing stub was subjected to sputter coating with a mixture of platinum and palladium until a thickness of 5 nm was achieved to improve overall conductivity. Finally, the sample was visualized under SEM  operated at 20 kV voltage.The shape and surface morphology of the optimized TAC-THQ-NLCs were characterized with the help of an electron microscope utilizing an instrument, transmission electron microscope (TEM), and scanning electron microscopy (SEM). For TEM analysis, one drop of 20 times diluted optimized formulation was mounted on a copper grid coated with copper. Thereafter, the formulation-bearing grid was stained negatively with phosphotungstic acid , and TAC-THQ-NLCs were recorded by utilizing the FT-IR instrument . The FT-IR spectrum of each sample was taken from the wavelength ranging from 4000 cmThe DSC curves of drugs (TAC and THQ), physical mixture , and lyophilized TAC-THQ-NLCs were recorded using a differential scanning calorimeter  to investigate the compatibility between the drugs and excipients, as well as the physical state of the drug within the lipid matrix of the NLCs. For conducting the DSC analysis, powder samples (2 mg) were taken into the aluminum pan and sealed. Then, the DSC curves of each sample were recorded from temperatures ranging between 20 °C to 200 °C. The temperature was calibrated by using indium (>99.95% purity) with a melting point of 156.6 °C. An empty aluminum pan was used as a reference. The nitrogen gas flow and rate of heating were set to 20 mL/min and 10 °C/min, respectively.An X-ray diffraction study was conducted to examine the crystalline or amorphous characteristics of drugs encapsulated in the matrix of the NLCs. The XRD diffractograms of TAC, THQ, GMS, physical mixture , mannitol, and lyophilized TAC-THQ-NLCs were recorded using an X-ray diffractometer . The samples were kept inside the sample holder of the XRD instrument, and the graph was recorded as 2θ values, ranging from 5° to 60° at a 10° scanning speed.w/w) under constant stirring, placed on a magnetic stirrer maintained at 1000 rpm until the gelling agent fully dispersed. Thereafter, the resulting mixture was left overnight for swelling, followed by neutralization with a triethanolamine addition (0.05% w/w) added dropwise to convert it into a TAC-THQ-NLC-gel (TAC-THQ-NG). A similar procedure was followed to prepare the TAC-THQ-suspension-gel (TAC-THQ-SG) [The application of TAC-THQ-NLCs on the skin is difficult due to their insufficient viscosity. Therefore, the optimized TAC-THQ-NLCs were converted into gels with the addition of a gelling agent  .The physical appearance, as well as the homogeneity of the prepared gels, was examined visually. The gel was analyzed for its pH using a pH meter  at room temperature. The drug content was calculated to assess the uniformity of drugs present in the prepared gels. For determining the TAC and THQ content in gels, 1 g of gels were dissolved in 10 mL methanol, filtered with 0.2 µ membrane filter, diluted appropriately, and then subjected to RP-HPLC .The viscosity of the optimized TAC-THQ-NG and TAC-THQ-SG were determined with the help of a Brookfield viscometer . The instrument was operated at 100 rpm for 160 s at room temperature . The sprThe texture analysis of the developed TAC-THQ-NG and TAC-THQ-SG were performed using the instrument TA.XTplus Texture Analyzer  at room temperature. Briefly, 100 g of each gel formulation (TAC-THQ-NG and TAC-THQ-SG) was placed into a 100 mL glass beaker and the surface was kept flat to avoid forming air bubbles. After that, different parameters of the gel, such as firmness, consistency, cohesiveness, and cohesion, were determined using the texture analyser software and represented in the form of a graph .w/v Tween 80 with the  phosphate buffer saline. For this study, the release media (200 mL) were taken into a vessel, in which the dialysis bag containing 1 gm (TAC-THQ-NG) and (TAC-THQ-SG) of the gel formulations were immersed. This assembly was positioned on a magnetic stirrer with a temperature of the plate regulated at 37 ± 1 °C and 100 rpm speed. The duration of the study was 24 h, and at every pre-decided time point, a sample volume of 2 mL was taken out, and the same volume of the fresh dissolution media was replaced. Thereafter, withdrawn samples were passed through a 0.2 μ membrane filter before being analyzed using RP-HPLC for drug quantification. The data obtained were subjected to different kinetics models to determine the drug release mechanism from the TAC-THQ-NG [A dialysis bag  technique was followed to investigate the TAC and THQ release from the TAC-THQ-NG and TAC-THQ-SG. The dissolution media were prepared by mixing 0.5% C-THQ-NG .5 HaCaT cells per well were transferred for culturing into 96 well plates and placed in a CO2 incubator for 24 h. The culture media consisted of a DMEM medium containing fetal bovine serum  and antibiotic solution , and the temperature was maintained at 37 °C with CO2 (5%). Post incubation, various samples prepared at different concentrations (0.01–50 µg/mL) were added to the HaCaT cells and kept for incubation for 24 h. Subsequently, MTT dye at 250 µg/mL was mixed well and further subjected to incubation for 2 h. After that, the culture media were removed from each well, followed by the addition of 100 µL DMSO to solubilize the developed formazan crystals. Next, the plate was placed for 5 min on a shaker to solubilize the formazan crystals completely. Finally, absorbance at 570 nm wavelength was noted using a microplate reader . Cell viability was calculated from the absorbance data, and the results are reported as their corresponding IC50 values.The antiproliferative activity of the TAC-THQ-NG, TAC-THQ-SG, TAC-NLC-gel (TAC-NG), and TAC-suspension-gel (TAC-SG) was evaluated in vitro using an MTT assay on the HaCaT cells . For this assay, 1 × 10The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay method was used for the estimation of the antioxidant activity of the optimized formulation . Briefly2 dimension) was excised, hairs and subcutaneous fat were cleaned and then washed at least 3 times with phosphate buffer saline  and fixed on the Franz diffusion cell between the donor and acceptor compartment. In this experiment, PBS; pH 7.4, 0.5% Tween 80 were taken as the diffusion media; hence the receptor compartment was filled with 10 mL PBS. After that, Rhodamine B (0.03%)-loaded NLC-gel and pure Rhodamine B-based gel were spread separately on the abdominal skin of mice fixed on Franz diffusion cells. This assembly was placed on the top of an electronic magnetic stirrer fixed at 100 rpm speed for 24 h, maintaining the temperature at 37 ± 1 °C. Thereafter, the skin was taken and washed with ethanol, and slides of 0.5 µm thickness were prepared. Finally, the permeation depths of both samples were visualized under a confocal microscope  [The depth of the permeated formulations (TAC-THQ-NG and the TAC-THQ-SG) in the skin was analyzed by confocal laser scanning microscopy (CLSM). For conducting this experiment, the skin (1 cmGermany) ,60.The physicochemical stability of the TAC-THQ-NLCs and TAC-THQ-NG was evaluated at three different storage conditions i.e., 5 ± 3 °C (refrigerated temperature) and 25 ± 2 °C (room temperature) for a period of 3 months. The samples were cautiously transferred to a glass vial and placed for 3 months under different storage conditions. After 3 months, samples were evaluated for PS, PDI, and %EE for the TAC-THQ-NLCs whereas TAC-THQ-NG formulations were evaluated for changes in pH, drug content, and spreadability .p < 0.05.All experimental data are presented as mean ± standard deviation (SD), and GraphPad Prism 8 software version 8.3.0 was used for statistical analysis. One-way ANOVA was used to determine the level of significance of the data that was acquired, and then Tukey’s multiple comparison tests were used. A statistically significant difference was defined as